Vitamin B metabolism proteins

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a vitamin B6 metabolic enzyme. The invention also relates to the construction of a chimeric gene encoding all or a portion of the vitamin B6 metabolic enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the vitamin B6 metabolic enzyme in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/096,342, filed Aug. 12, 1998.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingproteins involved in vitamin B6 metabolism in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Vitamins are organic nutrients required in small quantities for avariety of biochemical functions. Vitamins, generally, can not besynthesized by the body and must therefore be supplied by the diet.Besides being water soluble, vitamins in the B complex have little incommon from the chemical point of view. Excess B vitamins are rarelyaccumulated or stored and thus must be provided regularly.

[0004] Vitamin B 1 (thiamin) and vitamin B6 (pyridoxine, pyridoxal, andpyridoxamine) are essential for animal nutrition. Vitamin B6 consists ofthree closely related pyridine derivatives: pyridoxine, pyridoxal, andpyridoxamine. All forms of vitamin B6 are absorbed from the intestine,but some hydrolysis of the phosphate esters occurs during digestion. Inthe presence of magnesium ions and using ATP as a cofactor, pyridoxalkinase (EC 2.7.1.35) converts pyridoxine to pyridoxine 5′-phosphate. Atleast two pyridoxal kinases involved in the salvage pathway of vitaminB6 biosynthesis have been identified in Escherichia coli (Yang et al.(1998) J. Bacteriol. 180:1814-1821). Pyridoxine 5′-phosphate isconverted to pyridoxal 5′-phosphate by the action of pyridoxamine5′-phosphate oxidase (EC 1.4.3.5). Pyridoxal 5′-phosphate is the majorform of B6 transported in plasma. Pyridoxal phosphate is used intransamination and is an integral part in the enzymes which breakdownglycogen. Studies in yeast and bacteria show that loss of functionperturbs amino acid, fatty acid, and sterol metabolism (Lam, H. M. andWinkler, M. E. (1992) Bacteriol. 174:6033-6045; Zhao and Winkler (1995)J. Bacteriol. 177:883-891; Loubbardi et al. (1995) J. Bacteriol. 177:1817-1823).

[0005] Thiamin and Vitamin B6 are present in almost all plant and animaltissues commonly used as foods, but the content is usually small.Accordingly, enzymes responsible for their biosynthesis are potentialtargets for future antibiotics, fungicides, and herbicides. Thus, adetailed understanding of the activation, structure, mechanism,kinetics, and substrate-binding properties of the vitamin B1 and vitaminB6 biosynthetic enzymes from plants (and other organisms) would aid inthe rational design of chemical or other kinds of herbicides. Isolationand purification of the enzymes from plants would provide a valuabletool for the in vitro screening of inhibitors of vitamin B1 and vitaminB6 biosynthesis.

SUMMARY OF THE INVENTION

[0006] The instant invention relates to isolated nucleic acid fragmentsencoding enzymes involved in vitamin B6 metabolism. Specifically, thisinvention concerns an isolated nucleic acid fragment encoding apyridoxal kinase or a pyridoxamine-phosphate oxidase and an isolatednucleic acid fragment that is substantially similar to an isolatednucleic acid fragment encoding a pyridoxal kinase or apyridoxamine-phosphate oxidase. In addition, this invention relates to anucleic acid fragment that is complementary to the nucleic acid fragmentencoding pyridoxal kinase or pyridoxamine-phosphate oxidase.

[0007] An additional embodiment of the instant invention pertains to apolypeptide encoding all or a substantial portion of a vitamin B6metabolic enzyme selected from the group consisting of pyridoxal kinaseand pyridoxamine-phosphate oxidase.

[0008] In another embodiment, the instant invention relates to achimeric gene encoding a pyridoxal kinase or a pyridoxamine-phosphateoxidase, or to a chimeric gene that comprises a nucleic acid fragmentthat is complementary to a nucleic acid fragment encoding a pyridoxalkinase or a pyridoxamine-phosphate oxidase, operably linked to suitableregulatory sequences, wherein expression of the chimeric gene results inproduction of levels of the encoded protein in a transformed host cellthat is altered (i.e., increased or decreased) from the level producedin an untransformed host cell.

[0009] In a further embodiment, the instant invention concerns atransformed host cell comprising in its genome a chimeric gene encodinga pyridoxal kinase or a pyridoxamine-phosphate oxidase, operably linkedto suitable regulatory sequences. Expression of the chimeric generesults in production of altered levels of the encoded protein in thetransformed host cell. The transformed host cell can be of eukaryotic orprokaryotic origin, and include cells derived from higher plants andmicroorganisms. The invention also includes transformed plants thatarise from transformed host cells of higher plants, and seeds derivedfrom such transformed plants.

[0010] An additional embodiment of the instant invention concerns amethod of altering the level of expression of a pyridoxal kinase or apyridoxamine-phosphate oxidase in a transformed host cell comprising: a)transforming a host cell with a chimeric gene comprising a nucleic acidfragment encoding a pyridoxal kinase or a pyridoxamine-phosphateoxidase; and b) growing the transformed host cell under conditions thatare suitable for expression of the chimeric gene wherein expression ofthe chimeric gene results in production of altered levels of pyridoxalkinase or pyridoxamine-phosphate oxidase in the transformed host cell.

[0011] An addition embodiment of the instant invention concerns a methodfor obtaining a nucleic acid fragment encoding all or a substantialportion of an amino acid sequence encoding a pyridoxal kinase or apyridoxamine-phosphate oxidase.

[0012] A further embodiment of the instant invention is a method forevaluating at least one compound for its ability to inhibit the activityof a pyridoxal kinase or a pyridoxamine-phosphate oxidase, the methodcomprising the steps of: (a) transforming a host cell with a chimericgene comprising a nucleic acid fragment encoding a pyridoxal kinase or apyridoxamine-phosphate oxidase, operably linked to suitable regulatorysequences; (b) growing the transformed host cell under conditions thatare suitable for expression of the chimeric gene wherein expression ofthe chimeric gene results in production of pyridoxal kinase orpyridoxamine-phosphate oxidase in the transformed host cell; (c)optionally purifying the pyridoxal kinase or the pyridoxamine-phosphateoxidase expressed by the transformed host cell; (d) treating thepyridoxal kinase or the pyridoxamine-phosphate oxidase with a compoundto be tested; and (e) comparing the activity of the pyridoxal kinase orthe pyridoxamine-phosphate oxidase that has been treated with a testcompound to the activity of an untreated pyridoxal kinase orpyridoxamine-phosphate oxidase, thereby selecting compounds withpotential for inhibitory activity.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0013] The invention can be more fully understood from the followingdetailed description and the accompanying Sequence Listing which form apart of this application.

[0014] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Vitamin B6 Metabolic EnzymesSEQ ID NO: Protein Clone Designation (Nucleotide) (Amino Acid) CornPyridoxal Contig of: 1 2 Kinase cc1.mn0001.h9 cr1n.pk0161.c12p0004.cb1hc14r p0038.crvae17r Rice Pyridoxal rlr6.pk0096.a8 3 4 KinaseSoybean Pyridoxal Contig of: 5 6 Kinase sgc6c.pk001.o1 srm.pk0038.g4Wheat Pyridoxal Contig of: 7 8 Kinase wdk4c.pk006.119 wl1n.pk0106.e8Corn Pyridoxamine- Contig of: 9 10  Phosphate Oxidase cbn10.pk0048.g12cpd1c.pk007.15 cr1n.pk0063.f3 csi1n.pk0050.f8 p0010.cbpcs54rp0072.comfr39r p0072.comfu19r Rice Pyridoxamine- Contig of: 11  12 Phosphate Oxidase rlr48.pk0022.b10 rr1.pk097.c18 Soybean Contig of: 13 14  Pyridoxamine- sfl1.pk0095.g3 Phosphate Oxidase sr1.pk0006.b5 Wheatwr1.pk0018.f9 15  16  Pyridoxamine- Phosphate Oxidase

[0015] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0016] In the context of this disclosure, a number of terms shall beutilized. As used herein, a “nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A nucleic acidfragment in the form of a polymer of DNA may be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

[0017] As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

[0018] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof.

[0019] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by nucleic acid fragments that do not share 100%sequence identity with the gene to be suppressed. Moreover, alterationsin a nucleic acid fragment which result in the production of achemically equivalent amino acid at a given site, but do not effect thefunctional properties of the encoded polypeptide, are well known in theart. Thus, a codon for the amino acid alanine, a hydrophobic amino acid,may be substituted by a codon encoding another less hydrophobic residue,such as glycine, or a more hydrophobic residue, such as valine, leucine,or isoleucine. Similarly, changes which result in substitution of onenegatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts.

[0020] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C.

[0021] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 80% identical to theamino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0022] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0023] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0024] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0025] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0026] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0027] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic nucleotide segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. Promoters which cause a nucleic acid fragmentto be expressed in most cell types at most times are commonly referredto as “constitutive promoters”. New promoters of various types useful inplant cells are constantly being discovered; numerous examples may befound in the compilation by Okamuro and Goldberg (1989) Biochemistry ofPlants 15:1-82. It is further recognized that since in most cases theexact boundaries of regulatory sequences have not been completelydefined, nucleic acid fragments of different lengths may have identicalpromoter activity.

[0028] The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225).

[0029] The “3′ non-coding sequences” refer to nucleotide sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting MRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the MRNAprecursor. The use of different 3′ non-coding sequences is exemplifiedby Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0030] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0031] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single nucleic acid fragment so thatthe function of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0032] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0033] “Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

[0034] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0035] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

[0036] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0037] Nucleic acid fragments encoding at least a portion of severalvitamin B6 metabolic enzymes have been isolated and identified bycomparison of random plant CDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

[0038] For example, genes encoding other pyridoxal kinases orpyridoxamine-phosphate oxidases, either as cDNAs or genomic DNAs, couldbe isolated directly by using all or a portion of the instant nucleicacid fragments as DNA hybridization probes to screen libraries from anydesired plant employing methodology well known to those skilled in theart. Specific oligonucleotide probes based upon the instant nucleic acidsequences can be designed and synthesized by methods known in the art(Maniatis). Moreover, the entire sequences can be used directly tosynthesize DNA probes by methods known to the skilled artisan such asrandom primer DNA labeling, nick translation, or end-labelingtechniques, or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length CDNA or genomic fragments under conditions ofappropriate stringency.

[0039] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Primers oriented in the 3′ and 5′ directions can be designed fromthe instant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)Science 243:217-220). Products generated by the 3′ and 5′ RACEprocedures can be combined to generate full-length cDNAs (Frohman andMartin (1989) Techniques 1:165).

[0040] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen CDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol. 36:1; Maniatis).

[0041] The nucleic acid fragments of the instant invention may be usedto create transgenic plants in which the disclosed polypeptides arepresent at higher or lower levels than normal or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering the level of active vitamin B1 or vitamin B6in those cells.

[0042] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

[0043] Plasmid vectors comprising the instant chimeric gene can then beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

[0044] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100: 1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

[0045] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0046] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0047] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppressiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different chimeric genes utilizing differentregulatory elements known to the skilled artisan. Once transgenic plantsare obtained by one of the methods described above, it will be necessaryto screen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds, and is notan inherent part of the invention. For example, one can screen bylooking for changes in gene expression by using antibodies specific forthe protein encoded by the gene being suppressed, or one could establishassays that specifically measure enzyme activity. A preferred methodwill be one which allows large numbers of samples to be processedrapidly, since it will be expected that a large number of transformantswill be negative for the desired phenotype.

[0048] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded vitamin B6 metabolic enzyme. An example of a vector for highlevel expression of the instant polypeptides in a bacterial host isprovided (Example 7).

[0049] Additionally, the instant polypeptides can be used as a targetsto facilitate design and/or identification of inhibitors of thoseenzymes that may be useful as herbicides. This is desirable because thepolypeptides described herein catalyze various steps in vitamin B6metabolism. Accordingly, inhibition of the activity of one or more ofthe enzymes described herein could lead to inhibition of plant growth.Thus, the instant polypeptides could be appropriate for new herbicidediscovery and design.

[0050] All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

[0051] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bematzky and Tanksley (1986) Plant Mol.Biol. Reporter 4(1):37-41. Numerous publications describe geneticmapping of specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0052] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0053] In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan et al. (1995) GenomeResearch 5:13-20), improvements in sensitivity may allow performance ofFISH mapping using shorter probes.

[0054] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 1 7:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0055] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

[0056] The present invention is further defined in the followingExamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions.

Example 1 Composition of cDNA Libraries: Isolation and Sequencing ofcDNA Clones

[0057] cDNA libraries representing mRNAs from various corn, rice,soybean and wheat tissues were prepared. The characteristics of thelibraries are described below. TABLE 2 cDNA Libraries from Corn, Rice,Soybean and Wheat Library Tissue Clone cbn10 Corn Developing Kernel(Embryo and cbn10.pk0048.g12 Endosperm); 10 Days After Pollination cc1Corn Undifferentiated Callus cc1.mn0001.h9 cpd1c Corn Treated withChemicals Related to cpd1c.pk007.15 Protein Kinases* cr1n Corn Root From7 Day Old Seedlings** cr1n.pk0063.f3 cr1n.pk0161.c12 csi1n Corn Silk**csi1n.pk0050.f8 p0004 Corn Immature Ear p0004.cb1hc14r p0010 Corn LogPhase Suspension Cells Treated p0010.cbpcs54r With A23187*** to InduceMass Apoptosis p0038 Corn V5-Stage**** Roots p0038.crvae17r p0072 CornMesocotyl 14 Days After Planting p0072.comfr39r Etiolated Seedlingp0072.comfu19r rlr48 Rice Leaf 15 Days After Germination, 48rlr48.pk0022.b10 Hours After Infection of Strain Magaporthe grisea4360-R-62 (AVR2-YAMO); Resistant rlr6 Rice Leaf 15 Days AfterGermination, 6 rlr6.pk0096.a8 Hours After Infection of Strain Magaporthegrisea 4360-R-62 (AVR2-YAMO); Resistant rr1 Rice Root of Two Week OldDeveloping rr1.pk097.c18 Seedling sfl1 Soybean Immature Flowersfl1.pk0095.g3 sgc6c Soybean Cotyledon 16-26 Days After sgc6c.pk001.o1Germination (all yellow) sr1 Soybean Root sr1.pk0006.b5 srm Soybean RootMeristem srm.pk0038.g4 wdk4c Wheat Developing Kernel, 21 Days Afterwdk4c.pk006.119 Anthesis wl1n Wheat Leaf From 7 Day Old Seedling**wl1.pk0106.e8 wr1 Wheat Root From 7 Day Old Seedling wr1.pk0018.f9

[0058] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

[0059] cDNA clones encoding vitamin B6 metabolic enzymes were identifiedby conducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 2:5:403-410; see also www.nebi.nlm.nih.gov/BLAST/)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) NatureGenetics 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a CDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the CDNA sequence and theBLAST “hit” represent homologous proteins.

Example 3 Characterization of CDNA Clones Encoding Pyridoxal Kinase

[0060] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the cDNAs topyridoxal kinase from Sus scrofa and Homo sapiens (NCBI GeneralIdentifier Nos. 2773404 and 4505701, respectively). Shown in Table 3 arethe BLAST results for individual ESTs (“EST”), or contigs assembled fromtwo or more ESTs (“Contig”): TABLE 3 BLAST Results for SequencesEncoding Polypeptides Homologous to Pyridoxal Kinase BLAST pLog ScoreClone Status 2773404 4505701 Contig of: Contig 76.15 75.22 cc1.mn0001.h9cr1n.pk0161.c12 p0004.cb1hc14r p0038.crvae17r rlr6.pk0096.a8 EST 11.1012.00 Contig of: Contig 63.10 60.70 sgc6c.pk001.o1 srm.pk0038.g4 Contigof: Contig 65.52 65.40 wdk4c.pk006.119 wl1n.pk0106.e8

[0061] The data in Table 4 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6 and8 and the Sus scrofa and Homo sapiens (NCBI General Identifier Nos.2773404 and 4505701, respectively). TABLE 4 Percent Identity of AminoAcid Sequences Deduced From the Nucleotide Sequences of cDNA ClonesEncoding Polypeptides Homologous to Pyridoxal Kinase Percent Identity toSEQ ID NO. 2773404 4505701 2 44.5 42.9 4 24.3 28.7 6 54.6 49.5 8 47.345.7

[0062] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant CDNA clones encode an entire corn, and a substantial portion ofa rice, a soybean and a wheat pyridoxal kinase. These sequencesrepresent the first corn, rice, soybean and wheat sequences encodingpyridoxal kinase.

Example 4 Characterization of cDNA Clones EncodingPyridoxamine-Phosphate Oxidase

[0063] The BLASTX search using the EST sequences from clones listed inTable 5 revealed similarity of the polypeptides encoded by the cDNAs topyridoxamine-phosphate oxidase from Synechocystis sp., Caenorhabditiselegans and Rattus norvegicus (NCBI General Identifier Nos. 3122599,3979940 and 3237304, respectively). Shown in Table 5 are the BLASTresults for individual ESTs (“EST”), contigs assembled from thesequences of the entire cDNA inserts comprising the indicated cDNAclones and an EST (“Contig*”), or contigs assembled from two or moreESTs (“Contig”): TABLE 5 BLAST Results for Sequences EncodingPolypeptides Homologous to Pyridoxamine-Phosphate Oxidase NCBI GeneralBLAST Clone Status Identifier No. pLog Score Contig of: Contig 312259955.10 cbn10.pk0048.g12 cpd1c.pk007.15 cr1n.pk0063.f3 csi1n.pk0050.f8p0010.cbpcs54r p0072.comfr39r p0072.comfu19r Contig of: Contig 3979940 8.30 rlr48.pk0022.b10 rr1.pk097.c18 Contig of: Contig 3237304 57.10sfl1.pk0095.g3:fis sr1.pk0006.b5 wr1.pk0018.f9 EST 3237304 37.40

[0064] The data in Table 6 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:10, 12, 14and 16 and the Rattus norvegicus sequence (NCBI General Identifier No.3237304). TABLE 6 Percent Identity of Amino Acid Sequences Deduced Fromthe Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologousto Pyridoxamine-Phosphate Oxidase Percent Identity SEQ ID NO. to 323730410 38.3 12 14.5 14 40.2 16 45.3

[0065] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of apyridoxamine-phosphate oxidase. These sequences represent the firstcorn, rice, soybean and wheat sequences encoding pyridoxamine-phosphateoxidase.

Example 5 Expression of Chimeric Genes in Monocot Cells

[0066] A chimeric gene comprising a cDNA encoding the instantpolypeptides in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNAfragment encoding the instant polypeptides, and the 10 kD zein 3′region.

[0067] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0068] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0069] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0070] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0071] Seven days after bombardment the tissue can be transferred to N6medium that contains gluphosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining gluphosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0072] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 6 Expression of Chimeric Genes in Dicot Cells

[0073] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by Hind III sites.

[0074] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0075] Soybean embroys may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0076] Soybean embryogenic suspension cultures can maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0077] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0078] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0079] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0080] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0081] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Expression of Chimeric Genes in Microbial Cells

[0082] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0083] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC).Buffer and agarose contain 10 μg/ml ethidium bromide for visualizationof the DNA fragment. The fragment can then be purified from the agarosegel by digestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0084] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. Coli strain BL21 (DE3) (Studier et al. (1986) J.Mol. Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25°. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

Example 8 Evaluating Compounds for Their Ability to Inhibit the Activityof Vitamin B6 Metabolic Enzymes

[0085] The polypeptides described herein may be produced using anynumber of methods known to those skilled in the art. Such methodsinclude, but are not limited to, expression in bacteria as described inExample 7, or expression in eukaryotic cell culture, in planta, andusing viral expression systems in suitably infected organisms or celllines. The instant polypeptides may be expressed either as mature formsof the proteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

[0086] Purification of the instant polypeptides, if desired, may utilizeany number of separation technologies familiar to those skilled in theart of protein purification. Examples of such methods include, but arenot limited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

[0087] Crude, partially purified or purified enzyme, either alone or asa fusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity. For example, assays for pyridoxal kinase andpyridoxamine-phosphate oxidase are presented by Merrill and Wang (1986)Methods Enzymol. 122:110-116.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 16 <210> SEQ ID NO 1<211> LENGTH: 933 <212> TYPE: DNA <213> ORGANISM: Zea mays <400>SEQUENCE: 1 atggcgcggc cgccgatcct atccgtcgcg ctgccgtctg acaccggccgtgtgctcagc 60 atccagtccc acaccgtcca ggggtatgtt ggcaacaaat cggccgtctttcccctgcag 120 ctccttggct ttgatgtgga tccaataaac tctgtacagt tttctaatcatacaggatac 180 ccaacattta gaggtcaggt tcttaatggc aaacagctct gggaccttattgaaggactg 240 gaggaaaatc agttgcttca ttatacccat ttattaacag gttatataggctcagtttcc 300 tttttagata ctgtgctaca agttgttgag aaattgcgat cagttaatcctgatcttgta 360 tatgtttgtg acccagttct aggtgatgaa ggaaaactat atgttcctcaggaggtaata 420 tctgtttatc aacagaaggt tgttccagtt gcttcaatgc ttacacctaaccaatttgaa 480 gttgaactac ttactggatt gaggatcacc tccgaagaag atggtttgacagcttgtaat 540 accctccaca gtgccggacc acagaaggtg gttataacta gtgctcttattgaaggtaag 600 ctgctcctta tcggaagtca caaaaaaaca gaggaacaac agccagaacaatttaagatt 660 gagataccaa agatacctgc atatttcacg ggaactggag atttgacaactgctctccta 720 ctaggatgga gtaataaata tcctgatagc ctcgagaaag cagcagaactggcagtttcc 780 agtttgcagg cacttctgaa aagaactgtg gaagactata aaatggccggcttcgaccca 840 tcgaccagca gcttagagat ccggttgatc caaagccagg acgagatccgaaacccaact 900 gttacatgca aggctgtgaa gtatggaagc tga 933 <210> SEQ ID NO2 <211> LENGTH: 310 <212> TYPE: PRT <213> ORGANISM: Zea mays <400>SEQUENCE: 2 Met Ala Arg Pro Pro Ile Leu Ser Val Ala Leu Pro Ser Asp ThrGly 1 5 10 15 Arg Val Leu Ser Ile Gln Ser His Thr Val Gln Gly Tyr ValGly Asn 20 25 30 Lys Ser Ala Val Phe Pro Leu Gln Leu Leu Gly Phe Asp ValAsp Pro 35 40 45 Ile Asn Ser Val Gln Phe Ser Asn His Thr Gly Tyr Pro ThrPhe Arg 50 55 60 Gly Gln Val Leu Asn Gly Lys Gln Leu Trp Asp Leu Ile GluGly Leu 65 70 75 80 Glu Glu Asn Gln Leu Leu His Tyr Thr His Leu Leu ThrGly Tyr Ile 85 90 95 Gly Ser Val Ser Phe Leu Asp Thr Val Leu Gln Val ValGlu Lys Leu 100 105 110 Arg Ser Val Asn Pro Asp Leu Val Tyr Val Cys AspPro Val Leu Gly 115 120 125 Asp Glu Gly Lys Leu Tyr Val Pro Gln Glu ValIle Ser Val Tyr Gln 130 135 140 Gln Lys Val Val Pro Val Ala Ser Met LeuThr Pro Asn Gln Phe Glu 145 150 155 160 Val Glu Leu Leu Thr Gly Leu ArgIle Thr Ser Glu Glu Asp Gly Leu 165 170 175 Thr Ala Cys Asn Thr Leu HisSer Ala Gly Pro Gln Lys Val Val Ile 180 185 190 Thr Ser Ala Leu Ile GluGly Lys Leu Leu Leu Ile Gly Ser His Lys 195 200 205 Lys Thr Glu Glu GlnGln Pro Glu Gln Phe Lys Ile Glu Ile Pro Lys 210 215 220 Ile Pro Ala TyrPhe Thr Gly Thr Gly Asp Leu Thr Thr Ala Leu Leu 225 230 235 240 Leu GlyTrp Ser Asn Lys Tyr Pro Asp Ser Leu Glu Lys Ala Ala Glu 245 250 255 LeuAla Val Ser Ser Leu Gln Ala Leu Leu Lys Arg Thr Val Glu Asp 260 265 270Tyr Lys Met Ala Gly Phe Asp Pro Ser Thr Ser Ser Leu Glu Ile Arg 275 280285 Leu Ile Gln Ser Gln Asp Glu Ile Arg Asn Pro Thr Val Thr Cys Lys 290295 300 Ala Val Lys Tyr Gly Ser 305 310 <210> SEQ ID NO 3 <211> LENGTH:413 <212> TYPE: DNA <213> ORGANISM: Oryza sativa <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (380) <221> NAME/KEY: unsure <222>LOCATION: (384) <221> NAME/KEY: unsure <222> LOCATION: (388) <221>NAME/KEY: unsure <222> LOCATION: (410) <400> SEQUENCE: 3 gtttaaacaagaagatggct tgaaagcttg caatgcgcta catagtgctg gaccgcgaaa 60 ggtggtaataactagtgcac ttattgaaga taagctgctc ctcattggaa gccacaaaaa 120 agcaaaggaacaaccaccag aacaatttaa gattgagata cccaagatac ctgcatattt 180 cacgggcactggagatttaa caactgccct tctactagga tggagtaata aataccctga 240 taaccttggagagggcgctg aactggcggt atccatttgc aaggcacccc taaggagaac 300 tgtggaagactataaaagac tgggtttgac cctccaacca acacctagag atccgcctgg 360 attcaaaaccaaggatgaan tccnaagncc caagatacat gcaagctgtn aaa 413 <210> SEQ ID NO 4<211> LENGTH: 136 <212> TYPE: PRT <213> ORGANISM: Oryza sativa <220>FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (127)..(128)..(129)<400> SEQUENCE: 4 Phe Lys Gln Glu Asp Gly Leu Lys Ala Cys Asn Ala LeuHis Ser Ala 1 5 10 15 Gly Pro Arg Lys Val Val Ile Thr Ser Ala Leu IleGlu Asp Lys Leu 20 25 30 Leu Leu Ile Gly Ser His Lys Lys Ala Lys Glu GlnPro Pro Glu Gln 35 40 45 Phe Lys Ile Glu Ile Pro Lys Ile Pro Ala Tyr PheThr Gly Thr Gly 50 55 60 Asp Leu Thr Thr Ala Leu Leu Leu Gly Trp Ser AsnLys Tyr Pro Asp 65 70 75 80 Asn Leu Gly Glu Gly Ala Glu Leu Ala Val SerIle Cys Lys Ala Pro 85 90 95 Leu Arg Arg Thr Val Glu Asp Tyr Lys Arg LeuGly Leu Thr Leu Gln 100 105 110 Pro Thr Pro Arg Asp Pro Pro Gly Phe LysThr Lys Asp Glu Xaa Xaa 115 120 125 Xaa Pro Lys Ile His Ala Ser Cys 130135 <210> SEQ ID NO 5 <211> LENGTH: 812 <212> TYPE: DNA <213> ORGANISM:Glycine max <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (577)<221> NAME/KEY: unsure <222> LOCATION: (610) <221> NAME/KEY: unsure<222> LOCATION: (683) <221> NAME/KEY: unsure <222> LOCATION: (687) <221>NAME/KEY: unsure <222> LOCATION: (742) <221> NAME/KEY: unsure <222>LOCATION: (744) <221> NAME/KEY: unsure <222> LOCATION: (746) <221>NAME/KEY: unsure <222> LOCATION: (755) <221> NAME/KEY: unsure <222>LOCATION: (760) <221> NAME/KEY: unsure <222> LOCATION: (769) <221>NAME/KEY: unsure <222> LOCATION: (778) <221> NAME/KEY: unsure <222>LOCATION: (785)..(786) <221> NAME/KEY: unsure <222> LOCATION: (792)<221> NAME/KEY: unsure <222> LOCATION: (804) <400> SEQUENCE: 5gcacgaggag cattttccgg gcacgaaact cgaggaattc gcgcatggcg cctccaatcc 60tctcgctcgc tcttccctcg aacaccggtc gagttctcag cattcaatct cacaccgttc 120aggggtatgt tggtaataaa tccgctgtct tccctctgca actactggga tatgatgtcg 180atccaattaa ttccgtgcag ttttcgaatc atacaggata tccgacgttt aagggtcagg 240ttttgaatgg acagcaactc tgggatctaa tcgaaggcct tgaaggaaat gatttattgt 300tctatactca cttgctaaca ggttatattg gttcagagtc ttttctaaac actgtattgc 360aagttgtcag caaacttcgg tcaacaaacc caggtctttc gtatgtatgt gatccagtga 420tgggtgatga aggaaagctt tatgttcctc aagagctagt atcagtctat cgtgagaagg 480ttgttccagt agcttcaatg ttgactccca accagtttga agcagaacta ctgacaggct 540ttaggattca gtctgaagga catggccggg aggctgntag gcttctccat gcagctgggc 600cttcaaaggn cataattaca agtataaata tagacgggat tcttctcctc attggcagtc 660atccaaaaga aaagggagag ccncccngac aatttaagat tgttattcca aaaataacca 720gcttatttta cgggaacggg anancncatg actgnattcn tcttggttng agcataanta 780cccannacaa ancttgagaa tgcngcggaa ct 812 <210> SEQ ID NO 6 <211> LENGTH:196 <212> TYPE: PRT <213> ORGANISM: Glycine max <220> FEATURE: <221>NAME/KEY: UNSURE <222> LOCATION: (178) <221> NAME/KEY: UNSURE <222>LOCATION: (189) <400> SEQUENCE: 6 Met Ala Pro Pro Ile Leu Ser Leu AlaLeu Pro Ser Asn Thr Gly Arg 1 5 10 15 Val Leu Ser Ile Gln Ser His ThrVal Gln Gly Tyr Val Gly Asn Lys 20 25 30 Ser Ala Val Phe Pro Leu Gln LeuLeu Gly Tyr Asp Val Asp Pro Ile 35 40 45 Asn Ser Val Gln Phe Ser Asn HisThr Gly Tyr Pro Thr Phe Lys Gly 50 55 60 Gln Val Leu Asn Gly Gln Gln LeuTrp Asp Leu Ile Glu Gly Leu Glu 65 70 75 80 Gly Asn Asp Leu Leu Phe TyrThr His Leu Leu Thr Gly Tyr Ile Gly 85 90 95 Ser Glu Ser Phe Leu Asn ThrVal Leu Gln Val Val Ser Lys Leu Arg 100 105 110 Ser Thr Asn Pro Gly LeuSer Tyr Val Cys Asp Pro Val Met Gly Asp 115 120 125 Glu Gly Lys Leu TyrVal Pro Gln Glu Leu Val Ser Val Tyr Arg Glu 130 135 140 Lys Val Val ProVal Ala Ser Met Leu Thr Pro Asn Gln Phe Glu Ala 145 150 155 160 Glu LeuLeu Thr Gly Phe Arg Ile Gln Ser Glu Gly His Gly Arg Glu 165 170 175 AlaXaa Arg Leu Leu His Ala Ala Gly Pro Ser Lys Xaa Ile Ile Thr 180 185 190Ser Ile Asn Ile 195 <210> SEQ ID NO 7 <211> LENGTH: 773 <212> TYPE: DNA<213> ORGANISM: Triticum aestivum <400> SEQUENCE: 7 atggcgcggccgccgatcct atccgtcgcg ctgccgtctg acaccggccg tgtgctcagc 60 atccagtcccacaccgtcca ggggtatgtt ggcaacaaat cggccgtctt tcccctgcag 120 ctccttggctttgatgtgga tccaataaac tctgtacagt tttctaatca tacaggatac 180 ccaacatttagagggtcagt tcttaatggc aaacagctct gggaacttat tgaaggactg 240 gaggaaaatcagctgcttca ttatacccat ttattaacag gttatatagg ctcagtttcc 300 tttttagatactgtgctaca agttgttgag aaattgcgat cagttaatcc tgatcttgta 360 tatgtttgtgacccagttct aggtgatgaa ggaaaactat atgttcctca ggagctaata 420 tctgtttatcaacagaaggt tgttccagtt gcttcaatgc ttacacctaa ccaatttgaa 480 gttgaactacttactggatt gaggatcacc tccgaagaag atggtttgac agcttgtaat 540 accctccacagtgccggacc acagaaggtg gttataacta gtgctcttat tgaaggtaag 600 ctgctccttatcggaagtca caaaaaaaca gaggaacaac agccagaaca atttaagatt 660 gagataccaaagatacctgc atatttcacg ggaactggag atttgacaac tgctctccta 720 ctaggatggagtaataaata tcctgatatc ctcgaggggg ggccgtacca aat 773 <210> SEQ ID NO 8<211> LENGTH: 256 <212> TYPE: PRT <213> ORGANISM: Triticum aestivum<400> SEQUENCE: 8 Met Ala Arg Pro Pro Ile Leu Ser Val Ala Leu Pro SerAsp Thr Gly 1 5 10 15 Arg Val Leu Ser Ile Gln Ser His Thr Val Gln GlyTyr Val Gly Asn 20 25 30 Lys Ser Ala Val Phe Pro Leu Gln Leu Leu Gly PheAsp Val Asp Pro 35 40 45 Ile Asn Ser Val Gln Phe Ser Asn His Thr Gly TyrPro Thr Phe Arg 50 55 60 Gly Ser Val Leu Asn Gly Lys Gln Leu Trp Glu LeuIle Glu Gly Leu 65 70 75 80 Glu Glu Asn Gln Leu Leu His Tyr Thr His LeuLeu Thr Gly Tyr Ile 85 90 95 Gly Ser Val Ser Phe Leu Asp Thr Val Leu GlnVal Val Glu Lys Leu 100 105 110 Arg Ser Val Asn Pro Asp Leu Val Tyr ValCys Asp Pro Val Leu Gly 115 120 125 Asp Glu Gly Lys Leu Tyr Val Pro GlnGlu Leu Ile Ser Val Tyr Gln 130 135 140 Gln Lys Val Val Pro Val Ala SerMet Leu Thr Pro Asn Gln Phe Glu 145 150 155 160 Val Glu Leu Leu Thr GlyLeu Arg Ile Thr Ser Glu Glu Asp Gly Leu 165 170 175 Thr Ala Cys Asn ThrLeu His Ser Ala Gly Pro Gln Lys Val Val Ile 180 185 190 Thr Ser Ala LeuIle Glu Gly Lys Leu Leu Leu Ile Gly Ser His Lys 195 200 205 Lys Thr GluGlu Gln Gln Pro Glu Gln Phe Lys Ile Glu Ile Pro Lys 210 215 220 Ile ProAla Tyr Phe Thr Gly Thr Gly Asp Leu Thr Thr Ala Leu Leu 225 230 235 240Leu Gly Trp Ser Asn Lys Tyr Pro Asp Ile Leu Glu Gly Gly Tyr Gln 245 250255 <210> SEQ ID NO 9 <211> LENGTH: 828 <212> TYPE: DNA <213> ORGANISM:Zea mays <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (74)<400> SEQUENCE: 9 atgctggtgt cattgactgc acctaagctc tgtgcaaaaa agttcactggcccacaccat 60 tttcttgggg gaangtttgt ccccccacct attttaaacc aattacgggacttcagctcc 120 tcctttaccc tgggcacatc aatgtgtgtg agaattggaa aagctccatctgttgaaatt 180 tcatctctca gggagaacta tatttcccct gaacttcttg agagtcaagtgatgtctgat 240 ccatttgatc agttccttaa atggtttgat gaagcagtaa cagccggtcccggtctgcgt 300 gagcccaatg caatggcttt gacaactgcc aacaaggaag gaaaaccttcttcgaggatg 360 gttcttttaa agggagttga taaacaggga tttgtttggt atacaaattatggtagccgg 420 aaggcgcatg acttgtgtga aaaccctaac gcagcactcc ttttctactggaatgagatg 480 aaccgtcagg taagagttga agggtcagtt gagaaggttc cagaagctgaatcagataaa 540 tatttccaca gccgcccacg tggaagtcag cttggtgcca tagtcagcaagcagagtact 600 gtaattgctg gaagagaagt tcttcaacag gattacaaga aattggaacaaaaatattct 660 gatgggagct tgattccaaa acctgaatat tggggtggct acaaattgacaccgacactt 720 tttgagttct ggcaaggaca acagtctcga ctgcatgacc ggttacaatactcgcagaga 780 gaagtagatg ggagcacagt gtggcacatc gagaggttgt ccccttga 828<210> SEQ ID NO 10 <211> LENGTH: 275 <212> TYPE: PRT <213> ORGANISM: Zeamays <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (25) <400>SEQUENCE: 10 Met Leu Val Ser Leu Thr Ala Pro Lys Leu Cys Ala Lys Lys PheThr 1 5 10 15 Gly Pro His His Phe Leu Gly Gly Xaa Phe Val Pro Pro ProIle Leu 20 25 30 Asn Gln Leu Arg Asp Phe Ser Ser Ser Phe Thr Leu Gly ThrSer Met 35 40 45 Cys Val Arg Ile Gly Lys Ala Pro Ser Val Glu Ile Ser SerLeu Arg 50 55 60 Glu Asn Tyr Ile Ser Pro Glu Leu Leu Glu Ser Gln Val MetSer Asp 65 70 75 80 Pro Phe Asp Gln Phe Leu Lys Trp Phe Asp Glu Ala ValThr Ala Gly 85 90 95 Pro Gly Leu Arg Glu Pro Asn Ala Met Ala Leu Thr ThrAla Asn Lys 100 105 110 Glu Gly Lys Pro Ser Ser Arg Met Val Leu Leu LysGly Val Asp Lys 115 120 125 Gln Gly Phe Val Trp Tyr Thr Asn Tyr Gly SerArg Lys Ala His Asp 130 135 140 Leu Cys Glu Asn Pro Asn Ala Ala Leu LeuPhe Tyr Trp Asn Glu Met 145 150 155 160 Asn Arg Gln Val Arg Val Glu GlySer Val Glu Lys Val Pro Glu Ala 165 170 175 Glu Ser Asp Lys Tyr Phe HisSer Arg Pro Arg Gly Ser Gln Leu Gly 180 185 190 Ala Ile Val Ser Lys GlnSer Thr Val Ile Ala Gly Arg Glu Val Leu 195 200 205 Gln Gln Asp Tyr LysLys Leu Glu Gln Lys Tyr Ser Asp Gly Ser Leu 210 215 220 Ile Pro Lys ProGlu Tyr Trp Gly Gly Tyr Lys Leu Thr Pro Thr Leu 225 230 235 240 Phe GluPhe Trp Gln Gly Gln Gln Ser Arg Leu His Asp Arg Leu Gln 245 250 255 TyrSer Gln Arg Glu Val Asp Gly Ser Thr Val Trp His Ile Glu Arg 260 265 270Leu Ser Pro 275 <210> SEQ ID NO 11 <211> LENGTH: 555 <212> TYPE: DNA<213> ORGANISM: Oryza sativa <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (220) <221> NAME/KEY: unsure <222> LOCATION: (249) <221>NAME/KEY: unsure <222> LOCATION: (353) <221> NAME/KEY: unsure <222>LOCATION: (356) <221> NAME/KEY: unsure <222> LOCATION: (382) <221>NAME/KEY: unsure <222> LOCATION: (388) <221> NAME/KEY: unsure <222>LOCATION: (393) <221> NAME/KEY: unsure <222> LOCATION: (426) <221>NAME/KEY: unsure <222> LOCATION: (430) <221> NAME/KEY: unsure <222>LOCATION: (434) <221> NAME/KEY: unsure <222> LOCATION: (437) <221>NAME/KEY: unsure <222> LOCATION: (473) <221> NAME/KEY: unsure <222>LOCATION: (475) <221> NAME/KEY: unsure <222> LOCATION: (502) <221>NAME/KEY: unsure <222> LOCATION: (506) <221> NAME/KEY: unsure <222>LOCATION: (519) <221> NAME/KEY: unsure <222> LOCATION: (524) <221>NAME/KEY: unsure <222> LOCATION: (532) <221> NAME/KEY: unsure <222>LOCATION: (536)..(537) <221> NAME/KEY: unsure <222> LOCATION: (545)<221> NAME/KEY: unsure <222> LOCATION: (549) <221> NAME/KEY: unsure<222> LOCATION: (551) <400> SEQUENCE: 11 atgctggtat cattgactgcaccaaagctc tgtgcaaaaa aatttaccgg tccacaccat 60 tttcttgggg gtagatttgttcccccacct attgtgagca aatataagct tcatcttcct 120 ccatatcccg gtacctcaatgtgtgtgaga attggaaaag ctccatctgt tgacatttca 180 tctctaagaa gaaattacatctcccctgaa cttctcgagn aacaggtgat gcctgatcca 240 tttgataant tcgttagatggtttgatgaa ctgttacgct ggctacgtga accaaatgct 300 atggttaaca actccgataaggagggaaaa cttcgcaaag aatggccttt aanggngttg 360 ataaccacgg attttttgggancaattntg ganccaaaag gacatgatta cctgaaacca 420 aatgcngccn gttncantggaaggaataac ggcagtaaaa taaagtctgt canangtcca 480 gaaaagactg agatttcaaacnccanagga ataacttgng aatntcacac angcanncat 540 ctganggant ncagg 555<210> SEQ ID NO 12 <211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM:Oryza sativa <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (74)<221> NAME/KEY: UNSURE <222> LOCATION: (83) <400> SEQUENCE: 12 Met LeuVal Ser Leu Thr Ala Pro Lys Leu Cys Ala Lys Lys Phe Thr 1 5 10 15 GlyPro His His Phe Leu Gly Gly Arg Phe Val Pro Pro Pro Ile Val 20 25 30 SerLys Tyr Lys Leu His Leu Pro Pro Tyr Pro Gly Thr Ser Met Cys 35 40 45 ValArg Ile Gly Lys Ala Pro Ser Val Asp Ile Ser Ser Leu Arg Arg 50 55 60 AsnTyr Ile Ser Pro Glu Leu Leu Glu Xaa Gln Val Met Pro Asp Pro 65 70 75 80Phe Asp Xaa Phe Val Arg Trp Phe Asp Glu Leu Leu Arg Trp Leu Arg 85 90 95Glu Pro Asn Ala Met Val Asn Asn Ser Asp Lys Glu Gly Lys 100 105 110<210> SEQ ID NO 13 <211> LENGTH: 864 <212> TYPE: DNA <213> ORGANISM:Glycine max <400> SEQUENCE: 13 atgttgaaaa gggaagatgt tgatggtacaggcattaaac ctgatatgtt ggtttctttg 60 acagccccaa gattaggtgc aaagaagtttggtggtcctc accactttct aggaggtaga 120 tttgtcccac ctgctattgc agaaaaatataagcttatac ttccaccata tcctggaact 180 tccatgtgtg ttcgaattgg aaggcctccacgtattgata tctcagctct aagagagaac 240 tatatctctc cagaatttct tgaagagcaggtggaggctg acccttttaa tcagtttcat 300 aaatggttta atgatgcatt ggctgctggtttgaaggaac caaatgctat gtccttgtca 360 actgtaggga aggacggaaa accctcatcaagaatggtat tgctaaaagg cttggataag 420 gaaggatttg tgtggtacac aaactatgaaagtcgaaagg cacgtgaatt atctgaaaat 480 ccacgtgcat cacttctttt ttactgggatggtttaaacc ggcaggtacg ggtggaaggg 540 cctgttcaga aagtctctga tgaggaatcagaacagtatt tccatagccg ccctagaggt 600 agtcagattg gagcaatagt cagcaagcagagtactgtag tgccgggtag gcatgttctt 660 tatcaggagt acaaagagct ggaagaaaaatactctgatg gaagtttaat ccctaaacct 720 aagaactggg gtggatatag gctaacaccacaacttttcg agttttggca agggcagaaa 780 tctcgcttgc atgacaggtt gcaatatactccccatgaga tcaatggaca acggctgtgg 840 aaggttgacc ggttggctcc ttga 864<210> SEQ ID NO 14 <211> LENGTH: 287 <212> TYPE: PRT <213> ORGANISM:Glycine max <400> SEQUENCE: 14 Met Leu Lys Arg Glu Asp Val Asp Gly ThrGly Ile Lys Pro Asp Met 1 5 10 15 Leu Val Ser Leu Thr Ala Pro Arg LeuGly Ala Lys Lys Phe Gly Gly 20 25 30 Pro His His Phe Leu Gly Gly Arg PheVal Pro Pro Ala Ile Ala Glu 35 40 45 Lys Tyr Lys Leu Ile Leu Pro Pro TyrPro Gly Thr Ser Met Cys Val 50 55 60 Arg Ile Gly Arg Pro Pro Arg Ile AspIle Ser Ala Leu Arg Glu Asn 65 70 75 80 Tyr Ile Ser Pro Glu Phe Leu GluGlu Gln Val Glu Ala Asp Pro Phe 85 90 95 Asn Gln Phe His Lys Trp Phe AsnAsp Ala Leu Ala Ala Gly Leu Lys 100 105 110 Glu Pro Asn Ala Met Ser LeuSer Thr Val Gly Lys Asp Gly Lys Pro 115 120 125 Ser Ser Arg Met Val LeuLeu Lys Gly Leu Asp Lys Glu Gly Phe Val 130 135 140 Trp Tyr Thr Asn TyrGlu Ser Arg Lys Ala Arg Glu Leu Ser Glu Asn 145 150 155 160 Pro Arg AlaSer Leu Leu Phe Tyr Trp Asp Gly Leu Asn Arg Gln Val 165 170 175 Arg ValGlu Gly Pro Val Gln Lys Val Ser Asp Glu Glu Ser Glu Gln 180 185 190 TyrPhe His Ser Arg Pro Arg Gly Ser Gln Ile Gly Ala Ile Val Ser 195 200 205Lys Gln Ser Thr Val Val Pro Gly Arg His Val Leu Tyr Gln Glu Tyr 210 215220 Lys Glu Leu Glu Glu Lys Tyr Ser Asp Gly Ser Leu Ile Pro Lys Pro 225230 235 240 Lys Asn Trp Gly Gly Tyr Arg Leu Thr Pro Gln Leu Phe Glu PheTrp 245 250 255 Gln Gly Gln Lys Ser Arg Leu His Asp Arg Leu Gln Tyr ThrPro His 260 265 270 Glu Ile Asn Gly Gln Arg Leu Trp Lys Val Asp Arg LeuAla Pro 275 280 285 <210> SEQ ID NO 15 <211> LENGTH: 456 <212> TYPE: DNA<213> ORGANISM: Triticum aestivum <400> SEQUENCE: 15 cacgaggataagcagggatt cgtttggtac acaaattacg gtagccaaaa agcacatgat 60 ttatcggaaaattcaaatgc ggcacttctt ttctactgga atgagatgaa ccgacaggtt 120 agagtagaagggtcggttca gaaggtctca gaagaagaat ctgagaagta tttccacagc 180 cgcccacgtggaagtcagct tggtgcaatt gttagcaagc agagcactgt catttcttga 240 agagaagttctccaacaagc gtacaaggaa ttggagcaaa aatattctga cggtagcttc 300 atcccaaaacccgattactg gggtggctac aagttgacac caaatctttt tgagttctgg 360 caaggccagcagtctcgtct gcatgaccgg ctacagtatt cacagcgaga attaggtggg 420 agtacagaatggcacatcca aaggttgtcc ccttga 456 <210> SEQ ID NO 16 <211> LENGTH: 150<212> TYPE: PRT <213> ORGANISM: Triticum aestivum <400> SEQUENCE: 16 HisGlu Asp Lys Gln Gly Phe Val Trp Tyr Thr Asn Tyr Gly Ser Gln 1 5 10 15Lys Ala His Asp Leu Ser Glu Asn Ser Asn Ala Ala Leu Leu Phe Tyr 20 25 30Trp Asn Glu Met Asn Arg Gln Val Arg Val Glu Gly Ser Val Gln Lys 35 40 45Val Ser Glu Glu Glu Ser Glu Lys Tyr Phe His Ser Arg Pro Arg Gly 50 55 60Ser Gln Leu Gly Ala Ile Val Ser Lys Gln Ser Thr Val Ile Ser Arg 65 70 7580 Glu Val Leu Gln Gln Ala Tyr Lys Glu Leu Glu Gln Lys Tyr Ser Asp 85 9095 Gly Ser Phe Ile Pro Lys Pro Asp Tyr Trp Gly Gly Tyr Lys Leu Thr 100105 110 Pro Asn Leu Phe Glu Phe Trp Gln Gly Gln Gln Ser Arg Leu His Asp115 120 125 Arg Leu Gln Tyr Ser Gln Arg Glu Leu Gly Gly Ser Thr Glu TrpHis 130 135 140 Ile Gln Arg Leu Ser Pro 145 150

What is claimed is:
 1. An isolated nucleic acid fragment encoding apyridoxal kinase comprising a member selected from the group consistingof: (a) an isolated nucleic acid fragment comprising at least 400contiguous nucleotides wherein the nucleic acid fragment encodes anamino acid sequence that is at least 80% identical to the amino acidsequence set forth in a member selected from the group consisting of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8; (b) an isolatednucleic acid fragment that is complementary to (a).
 2. The isolatednucleic acid fragment of claim 1 wherein nucleic acid fragment is afunctional RNA.
 3. The isolated nucleic acid fragment of claim 1 whereinthe nucleotide sequence of the fragment comprises the sequence set forthin a member selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:3, SEQ ID NO:5 and SEQ ID NO:7.
 4. A chimeric gene comprising thenucleic acid fragment of claim 1 operably linked to suitable regulatorysequences.
 5. A transformed host cell comprising the chimeric gene ofclaim
 4. 6. An isolated nucleic acid fragment encoding apyridoxamine-phosphate oxidase comprising a member selected from thegroup consisting of: (a) an isolated nucleic acid fragment encoding anamino acid sequence that is at least 80% identical to the amino acidsequence set forth in a member selected from the group consisting of SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16; (b) an isolatednucleic acid fragment that is complementary to (a).
 7. The isolatednucleic acid fragment of claim 6 wherein nucleic acid fragment is afunctional RNA.
 8. The isolated nucleic acid fragment of claim 6 whereinthe nucleotide sequence of the fragment comprises the sequence set forthin a member selected from the group consisting of SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13 and SEQ ID NO:15.
 9. A chimeric gene comprising thenucleic acid fragment of claim 6 operably linked to suitable regulatorysequences.
 10. A transformed host cell comprising the chimeric gene ofclaim 9.